Summary/Abstract Current knowledge about megakaryocyte (MK) gene expression has focused largely on transcription, whereas translational processes are relatively poorly understood in the healthy state, and even less is known in disease states. Dysregulated microRNAs (miRs) play a critical role in inflammation and the pathogenesis of several human bone marrow diseases, and over-expression and knockout studies in mice have identified miRs that induce myeloproliferative disorders (MPDs). Nevertheless, the role of miRs in MK gene expression in hu- man health or disease remains poorly understood. This research will use in vivo, in vitro, biochemical and bio- informatic approaches to study miR regulation of normal and stress (IFN?)-induced MK gene expression. In our preliminary data, we identified miRs strongly associated with platelet count and function in 154 healthy do- nors. Knockdown and over-expression studies showed miR-125a-5p regulates proplatelet formation (PPF) and miR-15a-5p regulates GPVI-induced MK integrin activation in cultured MKs, and that both miRs are pre- dicted to target genes expressed in primary human bone marrow MKs. We hypothesize that these miRs regu- late in vivo megakaryocytopoiesis (MKpoiesis) and platelet production and function. Aim 1 will test this hy- pothesis in vivo using MK-specific ablation of miR-125a and miR-15a, as well as parenteral administration of anti-miRs, using assays of platelet production, Mkpoiesis, and GPVI-mediated platelet reactivity, thrombus formation and signaling. To understand how miRs function it is critical to know their mRNA targets. Until re- cently, the approaches for identifying miR targets have been based on computer algorithms with high false positive and false negative prediction rates, such that additional biochemical data is needed. We hypothesize that only Argonaute 2 (Ago)-bound miRs function in platelet production and function. Aim 2 will identify and characterize miR-125a-5p and miR-15a-5p mRNA targets in living MKs using an unbiased, transcriptome-wide approach called Ago-HITS-CLIP (high throughput sequencing of cross-linked argonate2 immunoprecipitates). Novel miR-mRNA pairs will be validated in cells and seed regions queried for functional SNPs using public GWAS data sets of cardiovascular disease. Our RNA-seq preliminary data demonstrate IFN?-stimulated cul- tured MKs regulates miR and mRNA expression, and we hypothesize that IFN?-regulated miRs are associated with IFN? responsiveness in MPDs. In Aim 3 we will perform Ago-HITS-CLIP on IFN?-treated hematopoietic progenitor and differentiating MKs and test for associations between IFN?-inducible miRs and hematologic re- sponse in IFN?-treated patients with MPDs. The major impact of this research will be to (1) provide insights into the roles of miR-125a-5p and miR-15a-5p in platelet production and function, (2) characterize active miRs in living normal and IFN?-stimulated MKs, and (3) lay groundwork for using miRs to enhance in vitro manufac- ture of platelets, as potential therapeutic targets for thrombocytopenia and thrombocytosis, and for mechanistic studies on pathogenic miRs in MPDs.